Enzymatic reduction of ketones in "micro-aqueous" media catalyzed by ADH-A from rhodococcus ruber

Article abstract of DOI:10.1021/ol070679c

Mono- and biphasic aqueous-organic solvent systems (50% v v^-1) as well as micro-aqueous organic systems (99% v v^-1) were successfully employed for the biocatalytic reduction of ketones catalyzed by alcohol dehydrogenase ADH-A from Rhodococcus ruber via hydrogen transfer. A clear correlation between the log P of the organic solvent and the enzyme activity-the higher, the better-was found. The use of organic solvents allowed highly stereoselective enzymatic carbonyl reductions at substrate concentrations close to 2.0 M.

Full text of DOI:10.1021/ol070679c

ORGANIC
LETTERS
2
007
Vol. 9, No. 11
163-2166
Enzymatic Reduction of Ketones in
Micro-aqueous” Media Catalyzed by
ADH-A from Rhodococcus ruber
2
“
†
‡
†
,†
Gonzalo de Gonzalo, Iv a´ n Lavandera, Kurt Faber, and Wolfgang Kroutil*
Department of Chemistry, Organic and Bioorganic Chemistry, UniVersity of Graz,
Heinrichstrasse 28, A-8010 Graz, Austria, and Research Centre Applied Biocatalysis,
Petergasse 14, A-8010 Graz, Austria c/o Department of Chemistry, Organic and
Bioorganic Chemistry, UniVersity of Graz, Heinrichstrasse 28, A-8010, Graz, Austria
wolfgang.kroutil@uni-graz.at
Received March 20, 2007
ABSTRACT
Mono- and biphasic aqueous
employed for the biocatalytic reduction of ketones catalyzed by alcohol dehydrogenase ADH-A from Rhodococcus ruber via hydrogen transfer.
A clear correlation between the log P of the organic solvent and the enzyme activity the higher, the better was found. The use of organic
solvents allowed highly stereoselective enzymatic carbonyl reductions at substrate concentrations close to 2.0 M.
−
organic solvent systems (50% v v-1) as well as micro-aqueous organic systems (99% v v-1) were successfully
s
s
The use of alcohol dehydrogenases (ADHs) for the stereo-
selective reduction of ketones and/or the oxidation of racemic
alcohols has gained increasing relevance during the past few
the majority of ketones of interest are highly hydrophobic,
and thus possess low solubility in aqueous media, which
leads to low substrate concentrations ranging from <5 to
10 mM. One of the traditional methods used to overcome
this drawback is the usage of organic solvents, widely
1
years, in particular for the synthesis of important intermedi-
2
ates of pharmaceuticals and bioactive compounds. However,
3
employed in the case of hydrolases. However, this technique
†
Department of Chemistry.
Research Centre Applied Biocatalysis.
has been scarcely applied for biocatalytic redox processes.
For instance, immobilized and pure horse liver alcohol
‡
(
1) (a) Zhang, J.; Witholt, B.; Li, Z. AdV. Synth. Catal. 2006, 348, 429.
4
5
(
b) Hanson, R. L.; Goldberg, S.; Goswami, A.; Tully, T. P.; Patel, R. N.
dehydrogenase (HLADH) and whole cells of baker’s yeast
AdV. Synth. Catal. 2005, 347, 1071. (c) Zhu, D.; Mukherjee, C.; Hua, L.
Tetrahedron: Asymmetry 2005, 16, 3275. (d) Kalaitzakis, D.; Rozzel, J.
D.; Kambourakis, S.; Smonou, I. Org. Lett. 2005, 7, 4799. (e) Kaluzna, I.
A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. Chem. Soc. 2004,
6
or Geotrichum candidum were shown to be active in hexane
(3) (a) Faber, K. Biotransformations in Organic Chemistry, 5th ed.;
Springer-Verlag: New York, 2004. (b) Carrea, G.; Riva, S. Angew. Chem.,
Int. Ed. 2000, 39, 2226.
(4) (a) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 8017. (b)
Grunwald, J.; Wirz, B.; Scollar, M. P.; Klibanov, A. M. J. Am. Chem. Soc.
1986, 108, 6732. (c) Jones, J. B.; Schwartz, H. M. Can. J. Chem. 1982, 60,
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(5) (a) Nakamura, K.; Inoue, K.; Ushio, K.; Oka, S.; Ohno, A. J. Org.
Chem. 1988, 53, 2589. (b) Nakamura, K.; Kondo, S.; Nakajima, N.; Ohno,
A. Tetrahedron 1995, 51, 687.
1
26, 12827. (f) Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr. Opin.
Chem. Biol. 2004, 8, 120. (g) Nakamura, K.; Matsuda, T. In Enzyme
Catalysis in Organic Synthesis; Drauz, K., Waldmann, H., Eds.; Wiley-
VCH: Weinheim, 2002; Vol. 3, pp 991. (h) Wills, M.; Hannedouche, J.
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In StereoselectiVe Biocatalysis; Patel, R. N., Ed.; Marcel Dekker: New York,
2
000; p 839.
2) Breuer, M.; Ditrich, K.; Habicher, T.; Hauer, B.; Kesseler, M.;
St u¨ rmer, R.; Zelinski, T. Angew. Chem., Int. Ed. 2004, 43, 788.
(
1
0.1021/ol070679c CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/01/2007

Figure 1. Effect of different organic solvents in aqueous-organic (50% v v-1) and micro-aqueous (99% v v-1) organic solvent systems
on the enzymatic reduction of 1a catalyzed by ADH-A from Rhodococcus ruber. Reactions were performed both with E. coli/ADH-A cells
(
30 U) and with recombinant enzyme (1.1 U) (t ) 24 h). log P (b) values are plotted to correlate enzyme activities with the hydrophobicity
of the organic solvents.
or benzene, respectively. In order to improve activities of
whole microbial cells in organic solvents, the cells had to
organic-solvent stability of ADH-A using the recombinant
enzymes and lyophilized whole cells of E. coli harboring
the overexpressed protein (E. coli/ADH-A).
6
,7
be immobilized. Subsequently, when purified enzymes
were employed as biocatalysts in biphasic systems, the
Two different solvent systems were employed for the
8
percentage of employed organic solvent was rather limited,
reduction of the model substrate 2-octanone (1a) to (S)-2-
probably due to the unstability of ADHs in this medium.
-1
octanol (1b): (a) organic cosolvent at 50% v v and (b)
-
1 13
We have recently demonstrated that alcohol dehydrogenase
micro-aqueous organic solvent at 99% v v .
9
ADH-A from Rhodococcus ruber DSM 44541 overex-
(a) Mono- or biphasic aqueous-organic solvent systems:
pressed in E. coli10 catalyzes the reduction of the ketones
As shown in Figure 1, lyophilized whole cells of E. coli/
ADH-A were fully active in all mono- or biphasic aqueous-
with simultaneous cofactor-recycling in a coupled substrate
11
approach using 2-propanol as hydrogen donor (Scheme 1).
-1
organic solvent systems at 50% v v . A moderate decrease
of conversion after 24 h was observed for recombinant
ADH-A, indicating probably a slight deactivation of the
enzyme. Detailed analysis of the solvent-effects showed a
continuous decrease of reaction rate upon gradual increase
of solvent concentration (detailed data are given in the
Supporting Information). In all cases, the stereoselectivity
of the enzyme remained completely intact leading to enan-
tiopure (S)-1b, emphasizing the robustness of the enzyme.
Scheme 1. Enzymatic Reduction of 2-octanone Catalyzed by
ADH-A in Aqueous-Organic and Micro-aqueous Media
Employing a Coupled Substrate Approach
(8) (a) Hu, J.; Xu, Y. Biotechnol. Lett. 2006, 28, 1115. (b) Lu, J.;
Lazzaroni, M. J.; Hallett, J. P.; Bommarius, A. S.; Liotta, C. L.; Eckert, C.
A. Ind. Eng. Chem. Res. 2004, 43, 1586. (c) Gr o¨ ger, H.; Hummel, W.;
Buchholz, S.; Drauz, K.; Van Nguyen, T.; Rollmann, C.; H u¨ sken, H.;
Abokitse, K. Org. Lett. 2003, 5, 173.
(9) (a) Poessl, T. M.; Kosjek, B.; Ellmer, U.; Gruber, C. C.; Edegger,
K.; Faber, K.; Hildebrant, P.; Bornscheuer, U. T.; Kroutil, W. AdV. Synth.
Catal. 2005, 347, 1827. (b) Stampfer, W.; Edegger, K.; Kosjek, B.; Faber,
K.; Kroutil, W. AdV. Synth. Catal. 2004, 346, 57. (c) Stampfer, W.; Kosjek,
B.; Faber, K.; Kroutil, W. J. Org. Chem. 2003, 68, 402. (d) Stampfer, W.;
Kosjek, B.; Moitzi, C.; Kroutil, W.; Faber, K. Angew. Chem., Int. Ed. 2002,
This enzyme showed impressive operational stability in the
presence of high concentrations of 2-propanol (up to 80% v
v ). Encouraged by these results, we decided to study the
4
1, 1014.
(10) Edegger, K.; Gruber, C. C.; Poessl, T. M.; Wallner, S. R.; Lavandera,
-1
12
I.; Faber, K.; Niehaus, F.; Eck, J.; Oehrlein, R.; Hafner, A.; Kroutil, W.
Chem. Commun. 2006, 2402.
(
11) Kosjek, B.; Stampfer, W.; Pogorevc, W.; Faber, K.; Kroutil, W.
(
6) Nakamura, K.; Inoue, Y.; Matsuda, T.; Misawa, T. J. Chem. Soc.,
Perkin Trans. 1 1999, 2397.
7) (a) De Temi n˜ o, D. M.-R.; Hartmeier, W.; Ansorge-Schumacher, M.
Biotechnol. Bioeng. 2004, 86, 55.
(12) Stampfer, W.; Kosjek, B.; Kroutil, W.; Faber, K. Biotechnol. Bioeng.
2003, 81, 865.
(
B. Enz. Microb. Technol. 2005, 36, 3. (b) Gervais, T. R.; Carta, G.; Gainer,
J. L. Biotechnol. Prog. 2003, 19, 389. (c) Luo, D.-H.; Zong, M.-H.; Xu,
J.-H. J. Mol. Catal. B: Enzym. 2003, 24-25, 83.
(13) Definition of “micro-aqueous”: Micro-aqueous solvent systems are
“monophasic” and are generally composed of e1 % water and g99%
organic solvent, see: Yamane, T. Biocatalysis 1988, 2, 1.
2164
Org. Lett., Vol. 9, No. 11, 2007

3
(
b) For micro-aqueous organic solvent systems at 99% v
filtration. Thus, the feasibility of recycling E. coli/ADH-A
-1
v , the picture drastically changed (Figure 1): water-
miscible solvents (DMSO, acetonitrile, 1,4-dioxane, EtOH)
led to complete deactivation of E. coli/ADH-A and recom-
binant ADH-A. Marginal activity was retained in 2-propanol,
which also acts as cosubstrate. In contrast, water-immiscible
solvents proved to be more biocompatible: reaction rates
fully recovered with E. coli/ADH-A and even with cell-free
ADH-A, deactivation was modest.1 Overall, a clear cor-
relation between the hydrophobicity of the solvents (ex-
pressed as log P) and enzyme activity can be seen: whereas
hydrophilic (water-miscible) organic solvents (log P < 0)
lead to complete deactivation, high biocompatibility is found
for hydrophobic solvents (log P > 2), such as toluene,
cyclohexane and hexane.
was investigated for the reduction of 1a in 99% hexane and
toluene. Reusing E. coli/ADH-A afforded the same excellent
stereoselectivity during all the cycles, thus enantiopure
(S)-1b was obtained. It was also observed that the E. coli
cells showed activity until the fifth cycle, in which no
reaction was observed for both solvents any more, allowing
to recycle the biocatalyst during four times until its total
deactivation.
4
The flexibility of ADH-A (E. coli/ADH-A or in partially
purified form) concerning its substrate spectrum was dem-
onstrated in micro-aqueous hexane. As shown in Table 1, a
15
Table 1. ADH-A-Catalyzed Bioreduction of Ketones in
Micro-aqueous Hexane (99% v v
The influence of the substrate concentration (1a) on the
activity and stereoselectivity of E. coli/ADH-A cells was
studied in 99% v v hexane (Figure 2). Although apparent
-
1 a
)
-
1
E. coli/ADH-A
rec ADH-A
substrate
productb
time (h)
c (%)
time (h) c (%)
Figure 2. Effect of substrate concentration (1a) on conversion (b)
and space time yield (2), expressed as g of ketone consumed per
L of solution h of E. coli/ADH-A (dashed line, 24 h) and purified
2a
3a
4a
5a
6a
7a
8a
(S)-2b
42
40
30
30
24
32
24
67.3
96.3
14.4
>99
56.1
35.4
76.4
42
42
24
30
30
24
42
63.1
48.1
20.1
68.8
76.6
41.8
74.1
-1
c
(R)-3b
(S)-4b
(R)-5b
6b
ADH-A (solid line, 48 h) in micro-aqueous hexane (99% containing
1% buffer-NADH solution).
c
(S)-7b
(2S,5S)-8b
conversions were lower at elevated substrate concentrations,
d
the space time yield (expressed as g of 2-octanone 1a
a
For experimental details see the Supporting Information. b ee >99%.
-
1
consumed per L of solution h ) increased, reaching a
c
Switch in CIP priority. d Sole product (2S,5S)-hexanediol (8b).
-
1
maximum at 0.94 M (120 g L ). A similar substrate
concentration-conversion profile-albeit with a slightly re-
duced maximum-was obtained for recombinant ADH-A,
which exihibited a maximum space time yield at 1.42 M
set of aromatic, linear and cyclic ketones (2a-7a) was
reduced to the corresponding alcohols 2b-7b. Depending
on the steric requirements of the substrate, variable reaction
times were required to reach moderate or good conversions.
For instance, enantiopure (R)-3b, an important pharma-
-
1
substrate concentration 1a (180 g L ). Surprisingly, even
at concentrations close to 2.0 M, recombinant ADH-A
exhibited reasonable activity, which is an important step for
up-scaling. The stereoselectivity of the enzyme remained
completely unchanged at elevated substrate concentrations
and (S)-1b was obtained in enantiopure form.
16
intermediate, was obtained with 96% conversion in 99%
hexane after 40 h using E. coli/ADH-A. Finally, a diketone
(8a) was employed as substrate. Similar to the results
One of the major advantages of using micro-aqueous
organic solvents is the facilitated recovery of enzymes by
17
obtained in buffer, enantiopure (2S,5S)-2,5-hexanediol (8b)
was achieved as the sole product. We were pleased, that no
trace of the intermediate keto-alcohol was observed after 24
(
14) Detailed experiments showed that at least 0.5% v v-1 of water is
required for activity.
15) Logarithm of the partition coefficient of a given compound in a
(
(16) Hamada, H.; Miura, T.; Kumobayashi, H.; Matsuda, T.; Harada,
standard n-octanol/water two-phase system.
T.; Nakamura, K. Biotechnol. Lett. 2001, 23, 1603.
Org. Lett., Vol. 9, No. 11, 2007
2165

h (c 76%, E. coli/ADH-A) or 42 h (c 74%, recombinant
ADH-A). In each case, the newly generated sec-alcohol
moieties were formed with absolute specificity (ee >99%).
clear correlation between log P and the biocompatibililty of
the organic solventsthe higher, the betterswas observed,
which is in contrast to recent observations using ADHs.18
It has been shown in this study that the recently cloned
and overexpressed ADH-A can successfully be employed
for the highly enantioselective reduction of a broad set of
ketones employing nonconventional aqueous-organic sol-
vent systems. The exceptionally robust enzyme efficiently
worked in micro-aqueous media composed of 99% of a very
hydrophobic organic solvent and buffer. These solvent
systems allowed to employ high substrate concentrations of
Acknowledgment. G.d.G. thanks the International
Human Frontier Scientific Program Organization (HFSPO)
for financial support. I.L. is financed by the Research Centre
Applied Biocatalysis, and financial support by FFG and
Province of Styria is gratefully acknowledged.
Supporting Information Available: Experimental and
kinetic data as well as the achiral and chiral GC data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
∼
2.0 M, which represents a 10-fold improvement in com-
parison to previous examples. Furthermore, an interesting
8c
OL070679C
(
17) Edegger, K.; Stampfer, W.; Seisser, B.; Faber, K.; Mayer, S. F.;
(18) Filho, M. V.; Stillger, T.; M u¨ ller, M.; Liese, A.; Wandrey, C. Angew.
Chem., Int. Ed. 2003, 42, 2993.
Oehrlein, R.; Hafner, A.; Kroutil, W. Eur. J. Org. Chem. 2006, 1904.
2166
Org. Lett., Vol. 9, No. 11, 2007